Synthetic hybrid grafts for craniofacial reconstruction: Sustained gene delivery using a calcium phosphate bone mineral substitute

These experiments were performed to evaluate the efficacy of a biocompatible bone cement, Norian CRS, engineered as a hybrid graft for simultaneous bone matrix reconstruction and sustained, site-directed gene transfer using an adenoviral vector. Norian CRS was cured ex vivo by mixing a calcium source powder with a phosphate source solution to form a paste. To 1.0 ml of the cement was added 50 mul of a solution containing 1 x 10(8) plaque-forming units of a replication-deficient adenoviral vector containing a bacterial beta -galactosidase reporter gene (AdLacZ). In vitro, fragments of the hybrid Norian-AdLacZ construct were placed into 12-mum-pore culture plate inserts and cocultured with human fibroblasts. The same insert was transferred to a new well of fibroblasts every 48 hours for 30 days, and, after allowing 72 hours for gene expression, fibroblasts were examined for transgene expression by 5 bromo-4-chloro-3-indoyl-beta -D-galactosidase (X-gal) staining. In vivo, the Norian-AdLacZ hybrid was implanted into 10-mm frontal bone defects in 3-week-old piglets. The implant sites were harvested after 5 days and were examined for transgene expression by X-gal staining. X-gal staining of fibroblasts incubated with the hybrid Norian-AdLacZ construct was observed throughout the 30-day period. Transgene expression was also observed about the periphery of the calvarial defects treated with hybrid Norian-AdLacZ constructs. Thus, adenoviral vectors may be incorporated successfully into a synthetic calcium phosphate bone mineral substitute to provide effective, sustained local gene delivery.